Plating Arabidopsis Seeds
Ben Carter, 9/24/2014 Notes: The removal of the Sterilization Solution and subsequent steps must be performed in a laminar flow hood using sterile technique. '' '''Surface Sterilization' Do not sterilize more than ~12 seed stocks at a time as this results in an unacceptable lengthening of the time the seeds are exposed to the Sterilization Solution and will result in sickly seedlings. Instead, sterilize the seeds in waves of up to 12 per wave. Sterilized seeds can be left at room temperature for several hours or up to 24h at 4°C. # Transfer an appropriate amount of the desired seeds from the 1dram seed library vials into labeled 1.5mL microfuge tubes. The volume of seeds required for one plate of 144 seedlings is approximately equivalent to a grain of rice. # Pipette 600µL of Sterilization Solution into each seed tube. Invert three times. Immediately place tubes on a rocker for 5min. # Immediately remove the Sterilization Solution using a P1000 or a glass pipette and house vacuum. Pipette 1mL of sterile water into the tubes, making sure to disturb the seeds in the process. # Remove the water as before and replace with a fresh 1mL of sterile water. Repeat this process for a total of five washes with water. The seeds will expand as they imbibe water during the washes. Seed Plating # Remove the appropriate MS Media Plates from the 4°C refrigerator. Place them in the laminar flow hood to warm. Once warmed, label the plates with the seed stock number, genotype, media type, the date, and your initials. # If excess condensation is present on the plate lid, remove using a sterile glass pipette and house vacuum. # Ensure that the last wash has been removed from the seeds. Add 200-600µL of sterile 0.15% agar to each tube. The amount used will depend on the quantity of seeds: one plate worth of seeds requires approximately 200µL. # Optional: Use a flame and sterile forceps to pull off the end of a sterile glass pipette. Using the forceps, clip the end of the pulled pipette to create a smaller opening. This eases the process of depositing single seeds. # Using a sterile glass pipette and rubber bulb, suspend the seeds in the first tube even in the agar. Use very little pressure to draw up a small amount of seeds. Deposit the seeds one-by-one on the media. Every square on the plate should get four evenly spaced seeds (imagine the '4' face of a die). Once completed, cover the plate and place it aside to # Use 3M gas-permeable tape to seal the plates at the lid-base interface. Place the plates in a plate incubator to develop. Reagents Sterilization Solution * 6.67mL of sterile water * 3.33mL of bleach * 100µL of 10% Triton X-100 MS Media Plates (1L = twenty plates) * 4.44g of MS-Gamborg powder (Sigma catalog# M0404) * 0.5g of MES * 10g of sucrose * 100µL of 20mg/mL glycine * 100mg of myo-inositol * Dissolve in 900mL of Milli-Q water and adjust pH to 5.6 using potassium hydroxide. * Dilute to 1L and transfer to a 2L Erlenmeyer flask. Add 9g of Bacto agar and a stir bar. * Cover the opening with loose aluminum foil and autoclave for 60min on a liquid cycle. * Place the media in a 50°C water bath for 30min. Add the desired additives, if any. Stir vigorously on a stir plate for 2min. * Pour the media into square plates approximately half full in the laminar flow hood and leave overnight to solidify. Store at 4°C. MS Media Additives and 1000X Stock Concentrations * Kanamycin: 30mg/mL in water, filter sterilize * Glufosinate (Basta): 10mg/mL in water, filter sterilize * Sulfadiazine: 5.25mg/mL in methanol or DMSO * Hygromycin B: 30mg/mL in water, filter sterilize * Rifampicin: 25mg/mL in DMSO (or methanol with a few drops of NaOH) * Dexamethasone: 0.01 M in methanol * Uniconazole: 10-5M in methanol * Gibberellic acid: 10-3M in methanol Category:Protocols __NOEDITSECTION__ Category:Arabidopsis